The present invention relates to animal models of sepsis and methods of use thereof. In particular, the invention relates to methods of predicting survival time of animals with sepsis, and methods of screening test compounds for effectiveness in treating sepsis.
Sepsis, or illness caused by a severe infection, is the third leading cause of infectious death (Bone, et al., 1997). The majority of sepsis cases are caused by bacterial infections. Accordingly, a substantial amount of money and time has gone into the search for drugs effective to treat sepsis and/or eliminate the pathogens (e.g., bacteria) which cause sepsis.
Current animal models of sepsis, used primarily to evaluate the efficacy of compounds in treating sepsis, are typically xe2x80x9cdeath as an endpointxe2x80x9d models. In other words, the determination of whether an animal has succumbed to sepsis (or if a particular treatment was effective) is made by scoring whether the animal dies during the course of the experiment.
Such death-as-an-endpoint models have a number of disadvantages. For example, because the investigator must wait until animals die before useful data may be obtained from the study, the study takes longer than it would if such data could be obtained at a time before death of the animals. From an animal welfare standpoint, death-as-an-endpoint models are disadvantageous because the animals die directly from the disease or toxic compounds under study, and are thus more likely to suffer as compared with animals that are euthanized by humane means earlier in the study.
It would therefore be desirable to have a reliable method by which an investigator could accurately predict whether and when a particular animal is likely to die as a result of sepsis without actually waiting for the animal to do so. The present invention provides such a method, as well as related methods suitable for screening drugs effective to treat sepsis.
In one aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which model system a critical rate of pathogen load increase has been ascertained; (ii) infecting an experimental animal of the animal species with a dose of reporter-labeled pathogen of the pathogen species, where the dose is sufficient to result in a rate of pathogen load increase exceeding the critical rate; (iii) administering a test drug to the experimental animal; (iv) measuring the level of the reporter in the experimental animal; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to decrease the rate of pathogen load increase in the experimental animal below the critical rate of pathogen load increase.
In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.
In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.
In another aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which animal species (a) a time of onset of terminal sepsis in response to a selected dose of the pathogen species, and (b) a critical infection level of the pathogen species, have been ascertained; (ii) infecting an experimental animal of the animal species with a dose of reporter-labeled pathogen of the pathogen species, where the dose is sufficient to cause the onset of terminal sepsis in an untreated animal; (iii) administering a test drug to the experimental animal; (iv) measuring the level of the reporter in the experimental animal at a selected time after onset of terminal sepsis, where the level of reporter corresponds to the level of infection in the experimental animal; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to drop the level of infection below the critical infection level.
In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.
In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.
In another aspect, the invention includes a method for selecting a candidate drug for treating sepsis. The method includes the steps of: (i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis in the animal species, in which animal species a time of onset of terminal sepsis, in response to a selected dose of the pathogen species, has been ascertained; (ii) infecting experimental and control animals of the animal species with a reporter-labeled pathogen of the pathogen species; (iii) administering a test drug to the experimental animals; (iv) measuring the level of reporter in the experimental and the control animals at a selected time after onset of terminal sepsis; and (v) selecting the test drug as a candidate drug for treating sepsis if the test drug is effective to cause a statistically-significant reduction in the level of reporter in the experimental animals as compared with the control animals.
In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.
In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.
The invention further includes a method for predicting an expected time of death of an experimental animal in a model system of sepsis. The method includes the steps of:
(i) selecting a model system of sepsis, the model system comprising an animal species and a pathogen species capable of causing sepsis, in which animal species a death expectation curve at a selected time has been constructed,
(ii) infecting an experimental animal of the animal species with a reporter-labeled pathogen of the pathogen species;
(iii) measuring the level of the reporter in the experimental animal at the selected time, and (iv) predicting the expected time of death from the level of the reporter and the death expectation curve.
In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.
In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.
The experiment also includes a method of predicting an expected time of death of an experimental animal in a model system of sepsis. The method includes the steps of (i) selecting a model system of sepsis, which model system comprises an animal species and a pathogen species capable of causing sepsis, and in which model system a correlation between actual time of death and a function of the rate of pathogen load increase has been established; (ii) infecting an experimental animal of the animal species with a reporter-labeled pathogen of the pathogen species; (iii) measuring the level of the reporter in the experimental animal at the selected time; and (iv) predicting the expected time of death from the level of the reporter and the correlation.
In one embodiment, the pathogen species is a bacterium species and the pathogen is a bacterium, e.g., a bacterium species selected from the group consisting of Enterococcus spp., Staphylococcus spp., Streptococcus spp., Enterobacteriacae family, Providencia spp. and Pseudomonas spp. An exemplary bacterium is a Pseudomonas spp.
In another embodiment, the animal species is a mammal, e.g., a rodent such as a mouse. In yet another embodiment, the reporter is light-emitting reporter, such as a luminescent reporter, e.g., a luciferase enzyme. In still another embodiment, the measuring is done using a photon detection device, such as an intensified CCD camera or a cooled CCD camera.